Reductases reduce unsaturated organic compounds, and have been used recently in an organic synthesis reaction for producing compounds that are used as active ingredients of medicaments or agrochemicals or intermediates thereof, especially for the production of optically active compounds.
According to the present invention, there is provided a modified reductase comprising an amino acid substitution(s) at certain amino acid residue(s) in a wild-type reductase amino acid sequence, thereby heat stability of the reductase is improved.
The present invention provides:                1) a reductase comprising                    i)an amino acid sequence of SEQ ID NO:1 having a substitution at amino acid position 245 or 271 or at both of the amino acid positions 245 and 271, or            ii)an amino acid sequence as defined in i) having further substitution, deletion, or addition of an amino acid or acids;                        2) a reductase according to 1) above, which comprises an amino acid sequence of SEQ ID NO:1 having a substitution at amino acid position 245 or 271 or at both of the amino acid positions 245 and 271;        3) a reductase according to 1) or 2) above, wherein said substitution is a single amino acid substitution at amino acid position 245 of the amino acid sequence of SEQ ID NO:1;        4) a reductase according to 1) or 2) above, wherein said substitution is a single amino acid substitution at amino acid position 271 of the amino acid sequence of SEQ ID NO:1;        5) a reductase according to 1) or 2) above, wherein the amino acids at positions 245 and 271 of the amino acid sequence of SEQ ID NO:1 are substituted with a same amino acid or different amino acids;        6) a reductase according to 3) or 5) above, wherein the amino acid at amino acid position 245 is substituted with arginine;        7) a reductase according to 4) or 5) above, wherein the amino acid at amino acid position 271 is substituted with aspartic acid;        8) a reductase according to 1) above, wherein the amino acid at amino acid position 245 of the amino acid sequence of SEQ ID NO:1 is substituted with arginine, and the amino acid at amino acid position 271 of the amino acid sequence of SEQ ID NO:1 is substituted with aspartic acid;        9) a polynucleotide sequence comprising a polynucleotide sequence encoding an amino acid sequence of the reductase of 1) or 2) above;        10) a vector comprising the polynucleotide of 9) above;        11) a transformant comprising the polynucleotide sequence of 9) above or the vector of 10) above;        12) a vector according to 10) above, which further comprises a polynucleotide sequence encoding an amino acid sequence of a protein capable of converting an NADP or an NAD into NADPH or NADH respectively;        13) a transformant of 11) above, which further comprises a polynucleotide sequence encoding the amino acid sequence of a protein capable of converting an NADP or NAD into NADPH or NADH respectively;        14) a method for producing (S)-halo-3-hydroxybutyrate ester, which comprises reacting 4-halo-3-oxobutyrate ester with the transformant of 11) or 13) above or a treated material thereof;        15) a method for modifying an enzyme, which comprises replacing at least one of the amino acids at positions 245 and 271 of the amino acid sequence of SEQ ID NO:1 respectively with another amino acid(s), thereby heat stability of said enzyme in the reduction reaction is improved; and        16) a method for producing a modified enzyme gene, which comprises replacing at least one codon corresponding to the amino acids at positions 245 and 271 of the amino acid sequence of SEQ ID NO:1, with another codon or codons corresponding to an amino acid(s), in a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1.        